The main research hypothesis in this grant proposal is that passive immunization with anti-prion monoclonal antibodies (Mabs) can effectively prevent neurological consequence resulting from extra- CNS prion infection. Prion diseases (prionoses) are transmissible, invariably fatal, neurodegenerative diseases associated with a conformational transformation of cellular prion protein PrPC into a toxic, infectious, and self-replicating PrPSC conformer for which no effective treatment is currently available. Accidental prion infection in humans may occur as a result of blood transfusion or organ transplant, ingestion of prion contaminated food, or use of prion contaminated surgical instruments. Following extra-CNS exposure, PrPSc replicates within the lymphoid organs for months to years before CNS invasion causes clinical symptoms. Therefore, prion infection in the pre-CNS stage can be targeted by therapeutics, which like Mabs are not required to have substantial blood-brain-barrier penetration. Revised aims of this grant proposal are as follows: Aim I will identify Mabs with strong therapeutic effect against human PrPSc replication, which can be humanized for clinical testing and application. Anti-PrP Mabs with strong therapeutic effect are unique. We have identified three Mabs: 6D11, 7H6, and 7A12 capable of permanent PrPSc abrogation in a cell culture model of murine prion infectivity with IC50%<1[unreadable]g/ml. In our preliminary studies, we have confirmed reactivity of these Mabs with human PrP and thereby we are planning to characterize their therapeutic effect using cell culture model of human prion infectivity which we are developing. Our testing will also include Mab 3F4, which strongly reacts with human PrPSc and has its antigen epitope located in the central portion of PrP sequence, a characteristic of other therapeutic Mabs. Aim II will address the fundamental question of whether passive immunization can prevent neurological consequence of prion infection. We have preliminary data to demonstrate that eight-week immunization with Mab-6D11 suppresses PrPSc replication in the lymphoid organs of extra-CNS infected wild type mice and the treatment extends the incubation period by ~40% and ameliorates brain pathology. The majority of experiments outlined in aim II will be carried out in murine models of prion infections using Mabs effective against murine PrPSc in cell culture models. If Mabs with therapeutic effect against human PrPSc replication are successfully identified in aim I, they shall be tested in transgenic mice expressing human PrP129M on murine PrP knock-out background which are susceptible to infection with human prions. The goal of revised aim III is to provide a mechanistic insight into therapeutic effect of Mabs. Aim III outlines a set of experiments investigating at what stage(s) of acquisition of scrapie form properties therapeutic anti-PrP Mabs interfere with PrPSc formation and whether they also facilitate PrPSc degradation. Furthermore, we will determine the cellular compartment(s) wherein Mabs activity occurs. The overall goal of this grant proposal is to provide a foundation for development of a passive immunization therapy for human prion infection. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a passive immunization approach to prevent humans accidentally exposed to prions from developing a universally fatal disease. Prionoses are transmissible neurodegenerative diseases for which there is no effective therapy. Moreover, prions are considered a potential bioterrorism agent.